Sex determination has been a favorite subject for reproductive physiologists since long. Sex determination is a process whereby the sex of the offspring is decided by sex chromosomes in mammals and by factors such as temperature in certain chordates such as reptiles. Sex assignment and determination in mammals is a method whereby the sex of the offspring is decided even before actual formation of the zygote by the fusion of an ovum with a spermatozoa bearing the sex chromosome of a particular sex and conditions are created such that the combination of chromosomes leading to the formation of a fetus of desired sex. Sex ratio is an indicator/measure devised to ascertain the proportion of males and females in a given population. There have been several attempts in the prior art to alter the sex ratio in mammals.
Some chordate species are known to be temperature dependent for sexual determination (TSD) (Bull, J. J 1980, Qrtly Rev Biol, 53, 3-21, 1980). In case of such animals, the sex of the offspring instead of being determined by sex chromosomes, is determined by the temperature at which egg is incubated. Further, there are certain species where application of certain hormones alters the sex of the offspring. Such species include certain reptiles and ratites. Such attempts to alter the sex are discussed and disclosed in U.S. Pat. Nos. 5,201,280 and 5,377,618.
However, in case of mammals, sex of the offspring is determined by random combinations of X- or Y-chromosome bearing sperms with an ovum always containing the X-chromosome and giving rise to a sex ratio of almost 50:50. In case of mammals also, some workers have tried to manipulate sex ratios. Manipulation of sex ratio gains prominence with the fact that mammals are an important family and success in altering the sex ratio in mammals, especially to the female side, has advantages in milk and meat producing species and in evolving livestock of better quality. This becomes doubly important in cross-breeding programs where 50% of the offsprings turn out to be of female sex and remaining 50% of male sex. The offsprings of the male sex are not generally favoured in the livestock industry. Altering the sex ratio, thereby leading to preferential production of females may make such cross-breeding programs a tremendous success. This also keeps the proportion of male populations of these species to the minimum extent as required for insemination purposes only.
In the light of these advantages, many workers have attempted to alter sex ratios throughout the last century. A number of attempts made about 1940 were based on the assumption that vaginal pH controlled the sex of the offspring. Schroeder V. (Physico-chemical methods of sex regulation of the progeny of mammals. Abstr in J. Hered 1941: 32:248) discussed efficacy of physico-chemical methods to regulate sex ratios. But conclusive evidence of efficacy of these methods was not found in careful scientific investigations (Salisbury and VanDemark: Physiology of reproduction and artificial insemination in cattle, 1961).
Later on, this approach of change in vaginal pH was deserted and reproductive physiologists concentrated on attempts to produce sexed semen. A sexed semen contains either X- or Y- bearing sperms in complete or accentuated concentrations, and which, when combined with ova, containing X-chromosome, either female (XX) or male (XY) offsprings are produced in complete or relatively greater proportions.
Several workers have carried on research on this subject and used different techniques to separate X- and Y- bearing sperms. (The sperms only contain X-Y-chromosomes, however, the applicant has referred the sperms as X- or Y- for convenience.) Lindahl in 1953 reported success in producing sexed semen in bulls (Counter-streaming of bull spermatozoa, Nature: 1956, 178: 491-92). Gordon M. J. (Scientific American, 199: 87-94, 1958) also discussed a method to control the sex ratio. This study represented yet another approach to produce sexed semen. Bhattacharaya et al (An Attempt to determine the sex ratio of calves by artificial insemination with spermatozoa separated by sedimentation, Nature 211: 863: 1966), were able to achieve a degree of success in producing sexed semen in bulls. Ericsson (Isolation of fractions rich in human Y-sperm, Nature 1973: 246:421) reported a method to get fractions richer in human Y-sperms. Gledhill (Control of mammalian sex by sexing the sperm: Fertil. Steril. 1983, 40(5): 572-74) and Corson et al (“Sex selection by sperm separation and insemination”, Fertil. Steril, 1984: 42:756) also reported new methods to produce sexed semen. However, no predictable and repeatable methodology could be evolved by these workers resulting in significant shift in sex ratio (Hunter, Reproduction of Farm Animals, 1982, 138-139), has also stated that despite all these attempts, modification of sex ratio still remains a mirage on the horizon. The reason behind this may be that sperms are haploid cells and haploid cells as distinct from diploid cells express a change in genetic constitution in surface characteristics, is still not clear (Hunter, 1982). Also, as pointed by Hafez (Reproduction in Farm Animals, fifth edition, 1987, 499), these attempts were hampered by the lack of laboratory tests to evaluate the degree of sperm separation.
Sex determination was reported also on a different line. Certain compounds, hormones, sera etc. were reported to have an effect on the altering of sex ratio. Bennett & Boyce, (Sex ratio in progeny of mice inseminated with sperm treated with HY antiserum, Nature, 246:308, 1973) reported that insemination with sperm treated with antisera to a Y-linked histocompatibility antigen produced 45.4% males compared with 53.4% for controls in mice. Barrat and Leger (J. Gyneiol. Obstet. Biol. Reprod, Paris, 8, 332, 1979) reported that administration of clomiphene citrate and/or gonadotropins resulted in 8.7% lowering of sex ratio. Beernik et al (“Factors influencing human sex ratio,” presented at the Annual meeting of American Fertility Society, 1984) reported similar results for humans. Sampson et al (“Gender After induction of Ovulation and artificial insemination” : Fertil. Steril. 40; pg 481, 1983) reported that with the induction of super ovulation, multiple births showed a marked skewness towards male births. Mitra & Chowdhary (Abstr in animal Breed, 58(4): No.2354, 1990) showed that glyceryl phosphorus choline diesterase activity of uterine fluid had an effect in altering the secondary sex ratio (i.e. at birth) in rats.
Thus, the prior art is replete with attempts to control the sex of mammalian offspring because the outcome of this research is valuable in a variety of economic conditions, for example, preferential birth of female calves in dairy herds would improve the rate of achieving superior animal strains by selective breeding. It would also be valuable medically where for example it is desired to prevent the birth of sons to mothers who are carriers of a genetic diseases which affect only males.
Each of the above attempts in the prior art represent different approaches towards sex assignment or production of off-springs of a desired sex. Recognizing the need to develop a simple and easy method for the assignment of sex or production of offsprings of significant number of females, the applicants conducted a thorough investigation on various chemicals capable of sex assignment and determination in offsprings. With the singular objective of increasing female population in livestock, the applicants screened a few chemicals and to their surprise found that a material essentially containing an acetyl group, such as vinegar, could be successfully used to achieve the above purpose. In addition, the applicant has arrived at a methodology to obtain female population, which is very cost-effective, easy to perform and does not involve in vitro treatment of sperms.